The plasma membranes of many mammalian cells are differentiated into biochemically and functionally distinct. This asymmetric arrangement of membrane components is best understood in the case of epithelial cells, whose surfaces are polarized into apical and basolateral domains that allow epithelia to serve as a selective barrier that regulates the interaction of tissues with the environment. Polarity in epithelial cells is generated by a series of molecular sorting events on the secretory and endocytic pathways that ensure the specific transport of newly synthesized, as well as internalized, membrane components to the appropriate plasma membrane domain. Despite the fundamental importance of these events, relatively little is known about the mechanisms by which endosomes and the Golgi complex distinguish between components destined for the apical and baolateral surfaces. During the previous grant period, we found that the sorting of membrane proteins in kidney epithelia, and probably other polarized cell types, is controlled by a novel set of distinct but generally distributed cytoplasmic domain determinants that specify transport to the basolateral plasma membrane. These determinants are often dependent on critical tyrosine or dileucine-containing motifs and thus bear some relationship with signals that specify localization at clathrin-coated pits. We also found that basolateral targeting determinants are dominant to, and hierarchically arranged with, a second generally distributed signal that specifies apical targeting: inactivation of the basolateral determinant invariably results in transport to the apical plasma membrane. In addition, the same basolateral signals are active in both the endocytic and secretory pathways, suggesting that similar or identical sorting strategies are used in both endosomes and the Golgi complex. Consequently, it seems likely that the sorting mechanisms used by epithelial cells may be widely conserved in different polarized cell types and throughout evolution. We plan to make use of this information and extend our work to elucidate the mechanism of polarized sorting at the cellular, biochemical, and molecular levels. Our Specific Aims include: (1) Characterize the structure, function, and distribution of basolateral sorting determinants; (2) Identify factors that regulate polarized sorting in intact cells; (3) Biochemically dissect the mechanism of sorting in endosomes through the use of cell-free assays that reconstitute receptor sorting and transport vesicle formation in vitro; (4) Identify and characterize cytosolic and/or coat proteins likely to be involved in endosome (or Golgi) function; (5) Characterize mammalian homologs of genes required for polarity during budding in yeast.